One of the major sites of action for cocaine and amphetamine in the brain is the dopamine transporter (DAT), a neuronal protein responsible for rapid re-uptake of dopamine after synaptic transmission. Cocaine and other psychostimulants act by inhibiting dopamine reuptake, which results in increased levels of synaptic dopamine and enhanced downstream neural activity. Some therapeutic agents such as methylphenidate also act at DAT, and dysfunctions in DAT activity may be related to dopaminergic disorders such as Parkinson s disease, depression, Attention Deficity Hyperactivity Disorder, and schizophrenia. It is currentlynot known if cocaine and other DAT inhibitors bind to the same or different sites on DAT or how they prevent transport. This basic lack of understanding of the molecular basis of the action of these compounds arises in part because so little is known about DAT structure and active sites, and is a major obstacle in the development of DAT-specific drugs that could be useful for treating psychostimulant abuse or other dopaminergic disorders. The goal of this study is to label DAT with irreversible analogs of cocaine and other uptake blockers and use proteolysis, epitope-specific immunoprecipitation, and mass spectrometry to identify the domains of the protein that interact with the ligands. The ligands to be developed will be modifications of previously-analyzed irreversible ligands whose sites of binding are known, and the patterns of incorporation of the new ligands, in conjunction with the previous findings, will lead to better understanding of the three-dimensional structure of DAT and the domains that contribute to antagonist and transport active sites. New ligands with distinct structures will also be examined. The long term objective of this research is to determine how DAT domains are spatially arranged to generate the binding sites for inhibitors, and to use this information to elucidate the molecular mechanisms underlying transport and transport inhibition. This knowledge will aid in the development of improved agents and therapeutic medications for drug abuse and other dopaminergic system disorders, and may be useful for generation of new agents for DAT imaging. The similarity of DAT to the norepinephrine and serotonin transporters presents the potential for these studies to be applicable to these other transporters.